Capillary electrophoresis or capillary zone electrophoresis, also referred to herein as CZE, is a relatively new entrant to the field of separation technologies. It shows great promise in several areas, especially in the biological chemicals arena. CZE is related to conventional electrophoresis, but shows much higher resolving ability, and much smaller sample sizes can be used. CZE separations involve introducing a buffered solution of analytes into a small-bore capillary column which is typically made of fused silica. A high-voltage differential is then established from one end of the column to the other. The surface silanol groups, because of their low pKa, are largely found in the Si--O-- form when the pH is greater than 2. The buffered solution flows with what is termed "electro-osmotic flow", because positive ions align with the negative Si--O-- groups, and are attracted to the ground end of the voltage differential. This flow sweeps the dissolved analytes along with it, each at a rate depending upon the analyte's charge-to-mass ratio. This charge-related flow of the analytes is termed the "electrophoretic mobility" of the analytes. Positively charged materials flow in the same direction as the electro-osmotic flow, but more slowly, neutrally charged molecules migrate at the same velocity and in the same direction as the electro-osmotic flow, and negatively charged materials tend to flow through the solution in the direction opposite the electro-osmotic flow. The electro-osmotic flow is frequently strong enough, however, that the net movement of negatively charged materials in the column is also in the same direction as the electro-osmotic flow. As a result, detectors may need to be placed at the end of the column opposite where simple charge flow would predict the analytes to elute. Overall separation is determined by the relative electrophoretic mobilities of the individual analytes. These mobilities can be varied by changes in the charge of the species, usually as a result of pH adjustment. The method is ascribed to J. Jorgenson and the initial publications are Analytical Chem., vol. 53, pp. 1298-1302 and J. Chrom., vol. 218, p. 209, both from 1981. The key discovery was that, by reducing the cross-sectional area of the column through use of microcapillary columns, the current density within the column could be reduced, thereby reducing the heating of the sample, which causes several problems.
Neutral analytes can also be separated by a variation of CZE termed micellar electrokinetic capillary chromatography, also referred to herein as MECC. In this technique a surfactant, usually sodium dodecyl sulfate, is added to the solution of the neutral analytes. As the sodium dodecyl sulfate is negatively charged, it moves in the opposite direction to the electro-osmotic flow. Neutral analytes are swept along with the electro-osmotic flow and are separated based on their partitioning between the surfactant and the surrounding aqueous phase. This technique was introduced by Terabe (Anal. Chem. vol.57, pp. 834-841 (1985) and vol. 56, pp. 111-113, (1984)).
The CZE and MECC techniques show promise for the separation of macromolecules of biological interest, including proteins and DNA fragments. The use for proteins is severly limited, however, as they tend to be adsorbed strongly upon the silica surface, which causes broadening of eluting analyte peaks, or even the loss of an entire analyte. This problem was discussed by Jorgenson in Science, vol. 222, p. 266 (1983).
Several attacks upon this problem of analyte adsorption onto the silica have been tried in CZE separations. These have involved changes in the mobile phase and coatings for the columns. The strengths and, more importantly, the weaknesses, of each are outlined below.
Changes in the mobile phase have included using phases of high and low pH or high ionic strength, and including organic modifiers in the phase. All of these techniques work to some extent, but they introduce other problems. As the primary interaction between a protein analyte and the surface is a charge interaction between the positive sites on the protein and the negatively charged surface, the pH changes either eliminate the positive charge on the protein (at high pH) or reduce the negative charge on the surface (at low pH). Unfortunately, either condition may denature the protein. Organic modifiers work by further solubilizing the protein, but conditions must be carefully chosen to avoid desolubilizing the protein. The organic modifiers may also denature the protein. Using a mobile phase of high ionic strength works by swamping the solution with buffer cations; this blocks protein interactions with the surface by shielding it with cations. The problem here is that it also makes the liquid much more conductive, which increases current flow and thus increases the heat generated. This heating will degrade resolution, and may even boil the mobile phase, precluding the separation altogether.
Another approach to the problem is to coat the walls of the silica column with an inert coating. Several such coatings have been tried.
Hjerten in U.S. Pat. No. 4,680,201 and J. Chrom. vol. 347 pp. 191-198 (1985) reacted the glass column walls with CH.sub.2 .dbd.CHCO.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 Si(OMe).sub.3, then copolymerized this with acrylamide, and also coated the column walls with methylcellulose and crosslinked this coating with formaldehyde. Both of these coatings were intended to eliminate electro-osmotic flow. This prevent both CZE and MECC separations, as both depend upon the electro-osmotic flow for separating analytes. Additionally, the published results indicated that interactions were still occurring.
Similarly, McCormick reported, in Anal. Chem. vol. 60, pp. 2322-2328, a poly(vinylpyrrolidinone) coating; his examples utilized low pH and relatively high ionic strength, which introduce the problems of heating and protein denaturing described above.
Poppe, as disclosed in J. Chrom. vol. 471, pp. 429-436 (1989), reacted the glass column walls with (MeO).sub.3 Si(CH.sub.2).sub.3 OCH.sub.2 -epoxide followed by a reaction with poly(ethylene glycol). He found these to be unsuitable for use above pH 5, which severly limits the utility of separations using such coated columns. Schomburg, in J. High Res. Chrom. 13, pp. 145-147 (1990), reported a crosslinked poly(ethylene glycol) for use in MECC, but only small-molecule separations were disclosed; its suitability for protein separation was not demonstrated, and details of the column preparation were not disclosed.
Maltose and epoxydiol have also been reported as column coatings by Bruin in J. Chrom., vol. 480, pp. 339-349, (1989). The epoxydiol coating gave poorer results than the poly(ethylene glycol)-coated columns described above, and also operated over a very limited pH range of 3 to 5. The maltose column operated over a wider pH range, but with relatively poor efficiency, and produced poorly shaped peaks.
A polyethyleneimine column has also been reported by Regnier in J. Chrom., vol. 516, pp. 69-78 (1990). This column was prepared by coating the surface with polyethyleneimine and then crosslinking the polyimine with a diepoxide. This coating reduced the dependence of the electro-osmotic flow on the mobile-phase pH; it also tended to reverse the direction of the flow. This was, however, a simple, mechanical coating which was not bonded to the surface. The life of such a non-bonded coating tends to be significantly shorter than that of a bonded coating.
Swedberg disclosed coatings made from (MeO).sub.3 Si(CH.sub.2).sub.3 NH.sub.2 in European Patent Publication No. 354 984 and PCT International Publication No. WO 89/12225. This material was first reacted with the glass column wall. The European patent discloses use of the material either as-is, or following reaction with glutaraldehyde and subsequently with a protein, dipeptide, or "other amphoteric compound". This yields a layer in which the electro-osmotic flow can be controlled by pH. The International Application discloses reacting the amine coating with the acetyl chloride of a moiety containing a plurality of halogen atoms, preferably a pentafluoroaryl moiety, to yield a layer which does not interact with proteins. Examples in each of these patents showed good separations, although they all employed high ionic strengths.
Sepaniak disclosed, in Anal. Chem. vol. 59, pp. 1466-1470 (1987), the use of trimethylsilyl chloride to give a trimethyl silyl coating which he used in MECC. No examples of analyzed proteins were given. This coating lowers electro-osmotic flow.
All of the above coatings with the exception of the second Swedberg and the Sepaniak papers are hydrophilic coatings which are similar to the types of coatings used on silica gel for high-performance liquid chromatography (HPLC) purposes.
Yeung, in Anal. Chem. vol. 62, pp. 2178-2182 (1990), reported a conventional, crosslinked dimethylsilicone coating. This exhibited undesirably low electro-osmotic flow when used in normal CZE, but reasonable flow rates in MECC. Schomburg, above, also reported a silicone coating used for MECC which exhibited increased electro-osmotic flow, but this flow also appears to have been inadequate for good CZE separations.
Tsuda, in J. Chrom., vol. 248, pp. 241-247, (1982), reported reacting a column surface with octadecyl silane, but very little information was given on the performance of the column, and use with biomolecules was not disclosed.
Capillary columns for gas chromatography (GC) may also be treated to deactivate the column surface. These deactivation procedures are intended primarily to keep amines from interacting with the siliceous surface and causing the eluting peaks to tail in gas chromatographic separations; no reason existed heretofore for applying them to CZE columns. The GC column preparation generally employs a 2-step procedure. In the first step the surface is deactivated using one of following primary methods: 1) high-temperature reaction with a cyclic siloxane; 2) high temperature reaction with a polymeric silicone or disiloxane; 3) high-temperature reaction with a silicone polymer containing Si-H groups; 4) high-temperature reaction with a disilazane; 5) low-temperature reaction with a silyl chloride; and 6) low-temperature reaction with a trialkoxysilane. Following deactivation, the column is coated with a silicone polymer, and this polymer is crosslinked in situ with free-radical initiators such as peroxides or azo compounds.
Each of the deactivation methods for GC columns has been used successfully except the reactions with silyl chloride (5) and trialkoxysilane (6). Although at least some success has been achieved with the trialkoxysilane it is not a preferred method. This is, however, the method disclosed for the GC deactivations described above. The methods involving polymers and cyclic polysiloxanes are preferred, as they are either easier to control or more successfully create an inert surface.
Some of these same approaches are also used to prepare silica-based packings for HPLC. The silyl chloride and trialkoxysilane reactions are most frequently used, but recently the silyl hydride polymer reactions have also been used, as reported by Lee in Anal. Chem., vol. 62, p. 1379 (1990). For HPLC, the goal is not so much to deactivate the surface but to provide a surface functionality for analyte interaction.
An object of the present invention is to deactivate the fused-silica walls of the capillary column to reduce the adsorption of analytes upon the silica during CZE, MECC and other chromatographic separations. Other objects will be apparent from the following description of the invention.